This Merit Review application is based on our hypotheses that Fibroblast-Like Synoviocytes (FLS) from Rheumatoid Arthritis (RA) patients infected with Epstein - Barr virus (EBV) will have unique gene expression properties that alter the host immune response to EBV, and that these difference influences the risk for developing RA. These hypotheses is based on the following observations: All 12 of the FLS cell lines that we have evaluated, six from Rheumatoid Arthritis (RA) patients and six from Osteoarthritis (OA) disease controls, contained some cells infected with EBV. We discovered that FLS can be superinfected with EBV. We observed that RA (but not OA) genetic risk loci are enriched for DNA variants that are immunoprecipitated with Epstein-Barr Nuclear Antigen-2 (EBNA2) (Relative Risk (RR)=4.5, Bonferroni corrected probability (Pc)=2.4x10-14). Preliminary data showed differences between RA and OA in the expression of the genes controlled by elements in RA risk loci. These observations and the 792 publications in Pubmed, often contradictory, present many intriguing relationships between EBV and RA. In our view these observations are consistent with EBV being involved in the pathogenesis of RA. We plan three initiatives with this DVA Merit Award project. First, we will characterize the virus infection in FLS (Aim 1) by: assessing the frequency of EBV FLS infection, sequencing the EBV DNA in FLS, and comparing EBV gene expression in RA, OA, & normal (NL) FLS. We will test the hypothesis that FLS infection is common among the EBV infected human population. Second, we will explore the FLS response to EBV infection (Aim 2) by determining host gene expression with RNA-seq to assess EBV infected and secondarily influenced FLS from RA, OA, & NL subjects and by exploring allele specificity at RA loci in RA, OA, and NL FLS. Once experimental conditions are determined, single cell RNA-seq will characterize the cell population infected with EBV and the cells responding to EBV infection. These experiments will evaluate the hypotheses that RA FLS will have a different gene expression profile than the FLS from OA or NL subjects and that the expression quantitative trait loci (eQTL) will distinguish RA from OA and NL FLS. Third, we will assess the immune response to EBV infected FLS (Aim 3). Our hypothesis is that the host response to EBV infection of the FLS will differentiate RA from OA and NL subjects. In specific, our detailed working hypothesis and current model is: 1. That usually the OA & NL FLS are abortively infected with EBV, 2. That OA & NL EBV infected FLS exist at a minimal level, perhaps only expressing a few EBV latent genes, as seen in latently infected B cells, and 3. That EBV infected OA & NL FLS are usually quiescent to immune recognition. In contrast, in RA there is evidence that the early steps of the EBV lytic program are initiated with the expression of Early- Immediate EBV antigens. These changes in combination with other factors (e.g., anti-cyclic citrullinated peptide autoantibodies, possible microbiome changes, etc.) awaken the quiescent immune response and result in an overwhelming inflammatory response directed against the EBV infected RA FLS in a way that does not happen in the OA or NL synovium. As has been our experience in the previous submissions, the results obtained may contradict any of these ideas leading this work into a new conceptual and strategic direction. On the other hand, this work has the potential to provide mechanistic details of the currently unknown aspects of RA pathogenesis, which would become the basis for new therapeutic approaches and preventive strategies. Whether or not EBV is a component of RA pathogenesis, the studies proposed will provide insight into a previously unknown tissue specific site of and possible reservoir for a common chronic viral infection that infects the vast majority of human beings.